Research ArticleMOLECULAR BIOLOGY

Two replication fork remodeling pathways generate nuclease substrates for distinct fork protection factors

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Science Advances  13 Nov 2020:
Vol. 6, no. 46, eabc3598
DOI: 10.1126/sciadv.abc3598
  • Fig. 1 53BP1 protects replication forks from nascent strand degradation.

    (A and B) Fork protection assays were completed by incubating cells with CldU for 30 min, followed by IdU for 30 min, and then treating with 4 mM HU for 4 hours. The lengths of CldU- and IdU-labeled DNA tracks were measured after DNA fiber spreading, and the ratio of IdU/CldU fiber lengths is plotted. All graphs are representative of at least two experiments. P values were calculated using a Kruskal-Wallis test. n.s., not significant. (A) U2OS cells were transfected with four individual siRNAs targeting 53BP1 or nontargeting (NT) siRNA and processed 72 hours after transfection. Immunoblotting was used to examine knockdown. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (B) CRISPR-Cas9–edited 53BP1 KO U2OS cells were analyzed for fork protection and compared to WT U2OS cells. (C) Quantitation of the γH2AX nuclear intensity in EdU-positive cells from the indicated 53BP1 KO cell lines. Cells were labeled with EdU for 10 min, treated with 4 mM HU for 2 hours, fixed, and stained. Each data point represents intensity in each S-phase cell. Bars are the mean, and P values were derived from a Kruskal-Wallis test. (D) Cell proliferation of the indicated cell lines (mean ± SD from n = 3). (E) Colony formation assay after exposure to the indicated dose of ionizing radiation (IR) (mean ± SD from n = 3).

  • Fig. 2 SMARCAL1, ZRANB3, and HLTF do not generate the substrate for nascent strand degradation in 53BP1-deficient cells.

    Fork protection assays and analyses were completed as in Fig. 1. (A to C) The indicated siRNAs were transfected into WT U2OS or SMARCAL1, ZRANB3, and HLTF 3KO U2OS cell lines before analyzing fork protection. (D) U2OS cells transfected with 53BP1 siRNA were treated with olaparib as indicated before analyzing fork protection. (E) WT or 3KO U2OS cells were transfected with the indicated siRNAs before analyzing fork protection. Cells were treated with mirin (50 μM) where indicated.

  • Fig. 3 SMARCAL1, ZRANB3, and HLTF dependency distinguishes two classes of fork protection proteins.

    Fork protection assays and analyses were completed as in Fig. 1. (A) WT U2OS cells or (B) SMARCAL1, ZRANB3, and HLTF 3KO U2OS cells were transfected with the indicated siRNAs before analyzing fork protection. (C) Heatmap summarizing fork protection screening results. siRNA was used to silence BRCA2, FANCD2, ABRO1, FANCA, VHL, 53BP1, or ABRO1 in the indicated KO cells or cells cotransfected with the indicated siRNAs. Data in WT U2OS, SMARCAL1 KO, HLTF KO, and 3KO cell lines were n = 3 experiments in which three different clones of each of the KO cell lines were analyzed. The other data are from n = 2 experiments. The mean value was used to create the heatmap after normalizing each experiment to the nontargeting control set at a ratio of 1.0. Lower values (pink) indicate more nascent strand degradation.

  • Fig. 4 FBH1 helicase activity is needed to generate the fork protection substrate for 53BP1.

    (A to C) Fork protection assays and analyses were completed as in Fig. 1. (A) WT U2OS cells were transfected with the indicated siRNAs. (B) WT U2OS cells were transfected with the indicated siRNAs and transfected with expression vectors encoding WT, F-box mutant (FM), or helicase mutant (HM) FBH1 before performing the fork protection assay. Immunoblots for 53BP1 and GFP-FBH1 are shown. (C) FBH1 KO U2OS cells were complemented with empty vector (EV), WT, FM, or HM FBH1-expressing retroviruses before depleting 53BP1 with siRNA and analyzing fork protection. HA-FBH1 protein expression was examined by immunoblotting. (D) Quantitation of the γH2AX nuclear intensity in EdU-positive cells transfected with the indicated siRNAs. Bars represent the mean, and P values were derived from a Kruskal-Wallis test. (E) U2OS cells transfected with the indicated siRNAs were treated with HU for 48 hours after plating for individual colonies. Colony number was scored after 10 days (mean ± SD, n = 3).

  • Fig. 5 FBH1 is required for nascent strand degradation in 53BP1-, BOD1L-, VHL-, FANCA-, FANCC-, and FANCG-deficient cells.

    Fork protection assays were completed as in Fig. 1 after siRNA transfections in (A and B) WT U2OS or (C) SMARCAL1, ZRANB3, and HLTF 3KO U2OS cells. (D) Nontargeting (NT), FANCC, or FANCG siRNAs were transfected into WT, 3KO, or FBH1 KO U2OS cells before assessing fork protection.

  • Fig. 6 BRCA2 is needed to generate the nascent strand degradation substrate in the FBH1 pathway and when RAD51 is partially inactivated.

    Fork protection assays were completed as in Fig. 1 in (A) WT or SMARCAL1, ZRANB3, and HLTF 3KO U2OS cells; (B) BRCA2-deficient PEO1 cells; (C) BRCA2-deficient VU423 cells; or (D) DLD-1 BRCA2 KO cells transfected with the indicated siRNAs. (E) FBH1 and HLTF double KO U2OS cells were complemented with WT, F-box mutant (FM), or helicase mutant (HM) FBH1 proteins by retrovirus infection. 53BP1 and BRCA2 were then silenced with siRNA before performing the fork protection assay. An immunoblot shows the expression levels of the HA-tagged FBH1 proteins. (F) U2OS cells transfected with BRCA2 siRNA were processed for the fork protection assay as indicated. B02 (25 μM) was included during the HU treatment where indicated. (G) DLD-1 WT or BRCA2 KO cells were treated with 25 μM B02 and/or 50 μM mirin during the HU treatment and assessed for fork protection.

Supplementary Materials

  • Supplementary Materials

    Two replication fork remodeling pathways generate nuclease substrates for distinct fork protection factors

    W. Liu, A. Krishnamoorthy, R. Zhao, D. Cortez

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